Altered cord blood mitochondrial DNA content and prenatal exposure to arsenic metabolites in low-arsenic areas

While mitochondria are susceptible to environmental detriments, little is known about potential associations between arsenic metabolites and mitochondria DNA copy number (mtDNAcn). We attempted to examine whether arsenic metabolism in different trimesters was related to cord blood mtDNAcn alteration. We included 819 mother-newborn pairs embedded in an in-progress birth cohort survey performed from April 2014 to October 2016 in Wuhan, China. We determined maternal urinary arsenic species concentrations in different trimesters using HPLC-ICPMS. We decided on cord blood mtDNAcn using quantitative real-time polymerase chain reaction. In covariate-adjusted models, each two-fold increment of dimethylated arsenic (DMA) and total arsenic (TAs) in the 3rd trimester were related to 8.43% (95% CI: 1.13%, 16.26%) and 12.15% (95% CI:4.35%, 20.53%) increases in mtDNAcn, respectively. The dose-response trend with statistical significance was observed across tertiles of DMA and TAs in the 3rd trimester with mtDNAcn. These findings may prove the relationships between arsenic species and mitochondrial dysfunction.


Introduction
Arsenic widely occurs in nature, existing in organic and inorganic forms.Inorganic arsenic (iAs) is generally considered more toxic than organic forms.The World Health Organization has reported that iAs in the groundwater exists at high concentrations (> 10 µg/L) in many countries, including Bangladesh, China, India, Chile, and Mexico (Ravenscroft et al. 2009).Dietary intake has become an essential source of arsenic exposure because of its accumulation in rice, sh, and dairy products (Chain 2009).People from the USA and some European countries have been reported to be exposed to arsenic at levels up to 300 µg/day via food and beverages (European Food Safety 2014, Kurzius-Spencer et al. 2014).iAs could be methylated to organic forms recognized as monomethylated arsenic (MMA) and dimethylated arsenic (DMA).More highly methylated arsenicals could be excreted more readily through the urine, decreasing retention and decreased biological exposure, and toxicity (Vahter 2002).Nevertheless, the idea has received a challenge considering that methylated trivalent arsenic showed more toxicity than is (Stýblo et al. 2002).
Existing evidence has revealed arsenic contributes to adverse health outcomes (Ravenscroft et al. 2009).
Long-term arsenic exposure could increase the hazards of cancer, diabetes, cardiovascular disease, and so on (ATSDR 2016, Kuo et al. 2017).Arsenic species could cross the placenta (Concha et al. 1998, Hall et al. 2007), resulting in newborns' susceptibility to arsenic exposure (Farzan et al. 2013).Existing epidemiologic research has revealed that early life exposure to arsenic species was linked to unfavorable Mitochondria, an essential ingredient of eukaryotic cells, plays a prominent role in many biological processes, like reactive oxygen species (ROS) production, apoptosis, and lipid metabolism (Shadel &Horvath 2015, Wai &Langer 2016).Mitochondria's vulnerability to environmental toxicants might be partially ascribed to its proximity to ROS (Dan Dunn et al. 2015), inadequate repair capacity, de ciency in noncoding introns, and lack of defensive histones (Malik &Czajka 2013).Nevertheless, mitochondria could compensate for genomic insults by modifying their abundance, ultimately altering mitochondrial DNA copy number (mtDNAcn) (Lee &Wei 2000).Generally, mtDNAcn, re ecting the number of copies of the mitochondrial genome per nucleated cell, is a hopeful marker of mitochondrial malfunction (Malik &Czajka 2013).Despite the variation in mtDNAcn among various cell types, mtDNAcn in speci c tissues/cells could remain unchangeable due to its rigorous modulation.Accumulating epidemiological studies have associated unexpected mtDNAcn variation from oxidative stress or in ammation with chronic diseases (Hägg et al. 2021 We attempted to make up for earlier surveys by estimating the relationships between maternal urinary arsenic metabolites with cord blood mtDNAcn and con rming the crucial exposure windows.

Study population
The analysis population is a subset of the Wuhan Healthy Baby Cohort that has enrolled pregnancies at the rst prenatal examination [gestational week less than 16] from 2012 to 2019.Eligibility criteria included uency in Chinese, singleton pregnancy, and intent to reside in Wuhan throughout gestation.The present analysis was restricted to 819 mother-newborn pairs recruited between April 2014 and October 2016, with prenatal arsenic metabolism exposure data and neonatal mtDNAcn data available derived from earlier research (Wang et al. 2021).
Informed written consent was acquired from all participators at recruitment.The Huazhong University of Science Institutional review boards have authorized the research procedures.

Urine acquisition and arsenic metabolite quanti cation
Details of urinary arsenic metabolite quanti cation are depicted elsewhere (Wang et al. 2021).In short, midstream spot urine samples were gathered during scheduled antenatal visits and stored at -20°C.Arsenic species were quanti ed by isotope dilution high-performance liquid chromatography coupled with quadrupole inductively coupled plasma mass spectrometer (HPLC-ICPMS), including arsenobetaine (AsB), arsenite (As 3+ ), arsenate (As 5+ ), MMA and DMA.Information about quality assurance, including limits of detections (LODs), limits of quanti cations (LOQs), and intra-day and inter-day coe cients of variation, are the same as that published by Wang et al. (Wang et al. 2021).Speci c gravity (SG) was determined to standardize the urinary arsenic species levels.

Relative mtDNAcn measurement
Venous umbilical cord blood was collected in vacutainer tubes containing EDTA immediately after delivery.Before analysis, blood samples were reserved at -80°C.Relative mtDNAcn measurement was carried out according to a prior described method (Janssen Bram et al. 2012).Information about detailed procedures of quantitative real-time PCR is presented in the supporting materials.

Covariates
Maternal demographic data (e.g., maternal age, family annual income, education levels, passive smoking, and folic acid intake) were collected by trained personnel via semi-structured questionnaires.In addition, data on pregnancy (e.g., parity, neonate gender, and pregnancy complications) was retrieved from the medical records.Information about the classi cation of pre-pregnancy body mass index (BMI) is shown in Supplementary Methods.Secondhand smoke exposure throughout gestation was de ned as passive smoke.

Statistical analysis
We conducted descriptive statistics analysis to characterize the distributions of variables of interest.Arsenic metabolite levels lower than the LOD were replaced with LOD/√2.We used SG to adjust for the dilution to correct urine dilution, which might potentially affect the association of arsenic metabolites with mtDNAcn.The formula of SG normalization is exhibited in the Supplementary Methods.
Urinary arsenic metabolite levels and relative mtDNAcn were log2-transformed to alleviate the skewed distribution.We calculated intraclass correlation coe cients (ICCs) to evaluate the reproducibility of urinary arsenic species levels in the 1st, 2nd, and 3rd trimesters (Rosner 2011).We averaged the SGadjusted arsenic metabolite levels in different trimesters to generate individual-speci c averages throughout gestation-arsenic metabolites.Details about arsenic exposure indices are shown in the Supplementary Methods.
Potential nonlinear associations between arsenic species and cord blood mtDNAcn were examined using 3-knot restricted cubic splines.Except for iAs and mtDNAcn (P = 0.008), linear relationships were observed between DMA, MMA, TAs, and mtDNAcn (P > 0.05).Thus, a general linear model was performed to examine the potential connection of individual-speci c arsenic metabolite averages (continuous and in tertiles) across three trimesters with cord blood mtDNAcn.Also, generalized estimating equations (GEEs) were performed to assess the relationships of trimester-speci c exposures (both continuous and in tertiles) with mtDNAcn (Sánchez Brisa et al. 2011).Tertile cut-points originated from trimester-speci c determination, and we de ned the lowest tertile as the reference.In addition, we modeled the median value of every tertile to determine the linear trends of these relationships.We converted beta (β) into percent change (%Δ) of exposures to make the statistical models more interpretable (Barrera-Gómez &Basagaña 2015).Covariates considered for inclusion were those changed estimates (≥ 10%) in the relationships or those observed to be linked to mtDNAcn in previous studies were based on previous studies and on.The following variables were included as covariables: maternal age at enrollment, gestational age (weeks), pre-pregnancy BMI (< 18.5, 18.5-23.9,≥ 24 kg/m 2 ), parity history (nulliparous or not), infant sex (male or not), education level (< 9, 9-12, ≥ 12 years), smoking exposure (passive smoking or not), gestational diabetes mellitus (yes or not), and gestational hypertension disorders (yes or not).
To test the robustness of our results, we completed additional analyses by excluding participants with PTB, LBW, gestational diabetes, or hypertensive disorders.Software SAS (version 9.4, Institute Inc., USA) was used.

Characteristics
Table 1 exhibits the characteristics of the mother-newborn pairs.The mean maternal age at enrollment was 28.94 years.Approximately 68.74% of participating women had a normal pre-pregnancy weight, with an average pre-pregnancy BMI of 20.99 kg/m 2 .Of the mothers in the analysis sample, 27.11% of women reported exposure to secondhand smoke, 88.03% acquired a college education or above, and 80.59% were primiparous.The proportion of gestational diabetes and hypertension disorders was 8.06% and 2.93%, respectively.For newborns, 53.60% were male, and 3.42% were born preterm.

Relationships of maternal arsenic exposure with cord blood mtDNAcn
Figure 1 lists the relationships of average levels of individual arsenic metabolites across three trimesters with the cord blood mtDNAcn alternation (Supplementary Table 3).In crude models, one double increase in DMA and TAs levels was related to an 8.56% (95% CI: ).In the dose-response trend analysis, the positive association between TAs level and mtDNAcn remained signi cant (p trend = 0.0423), yet there wasn't a remarkable relationship between DMA and mtDNAcn (p trend = 0.0854).Sensitivity analyses didn't markedly alter our results (Supplementary Table 4).

Discussion
In this longitudinal cohort survey, we evaluated the associations of maternal urinary arsenic species levels with cord blood mtDNAcn.We observed that elevated cord blood mtDNAcn was linked to exposure to DMA and TAs in the third trimester, where signi cant dose-response associations were also found.
Arsenic widely exists on the earth, leading to the risk of environmental exposure.It can be converted to MMA and DMA in humans, and urinary arsenic species are acknowledged as a marker of arsenic exposure (Hayakawa et al. 2005) ).Our reported ICCs (0.17-0.39) for repeated urinary arsenic speciation measurements were low, revealing high variability over the three trimesters.The low ICCs might be partly due to the alteration in external exposure level of arsenic speciation or the woman's physiology attributed to the progression of pregnancy (Abduljalil et al. 2012).Given the poor reproducibility in this study, multiple or consecutive measurements along the pregnancy are desired to characterize arsenic speciation exposure during the whole pregnancy accurately.
The mtDNAcn, quantifying mitochondrial genome abundance, is a marker of mitochondrial function and oxidative stress (Castellani et 2008).Second, oxidative stress biomarkers in cord blood samples weren't determined, which were associated with mitochondrial function (Liu et al. 2003).Finally, all participants in this survey were Chinese, which limited external generalizability to other populations.Further surveys of different people are suggested to con rm our ndings.

Conclusion
In this prospective pre-birth cohort from Wuhan, China, DMA and TAs exposure in the third trimester was linked to elevated cord blood mtDNAcn.Our ndings provided epidemiological data on mtDNA as a target of metals and emphasized a possible complicated part of arsenic specie in pathways of disease.Additional surveys are demanded to shed light on the molecular pathways involved in cord blood mtDNA damage induced by maternal methylated arsenic exposure.

Declarations
Figure 1 Associations of average maternal arsenic concentrations with cord blood mtDNAcn (N=819).Models were adjusted for maternal age at enrollment, gestational age, pre-pregnancy BMI, parity history, infant sex, education level, passive smoking during pregnancy, hypertensive disorders in pregnancy, and gestational diabetes mellitus.
). Arsenic, widely distributed in nature, is recognized as hazardous to human health (Jomova &Valko 2011, Tian et al. 2023, Valko et al. 2005).Numerous epidemiological surveys have suggested that in-utero exposure to aluminum, manganese, and lead was related to elevated cord blood mtDNAcn (Kupsco et al. 2019, Liu et al. 2019, Smith Anna et al. 2021), while prenatal exposure to arsenic, magnesium, and thallium was linked to reduced cord blood mtDNAcn (Smith Anna et al. 2021, Song et al. 2020, Wu et al. 2019).Nevertheless, no published epidemiological surveys have investigated arsenic metabolism and mtDNAcn associations among susceptible populations (e.g., pregnant women and neonates).

Table 1
Descriptive characteristics of study participants included in this study (N = 819).
. The urinary arsenic speciation levels in this study were lower than those in Mexico (median of DMA, MMA, TAs were 20.6, 1.4, and 23.3 µg/L) (Laine Jessica et al. 2015) and Pabna (median of DMA, MMA, TAs were 50.2, 3.4 and 63.2 µg/L) pregnant women (Gao et al. 2019).Additionally, the urinary arsenic speciation levels in this study were higher than those of pregnant women in the INMA Project (geometric mean[GM] of DMA, MMA, iAs were 5.54, 0.28 and 0.27 µg/L) (Soler-Blasco et al. 2021), Oklahoma City (GM of DMA, MMA, iAs and TAs were 3.93, <LOD, 1.36 and 4.46 µg/L) (Chen et al. 2021), the PHIME-CROME project (GM of DMA, MMA, TAs were 2.67, 0.17 and 3.23 µg/L) (Stajnko et al. 2019) and the MADRES study (median of DMA, MMA, iAs and TAs were 4.4, 0.4, 0.5 and 5.4 µg/L) (Farzan et al. 2021).Different urinary arsenic speciation levels in other regions might result from variations in research participants, living environments, lifestyle behaviors, dietary intakes, and individual methylation e ciency.Urinary iAs re ect recently personal iAs absorbed dose, and unique iAs methylation patterns remain stable for 8-10 months (Steinmaus et al. 2005).Urinary arsenic concentration could be a surrogate for exogenous multiple exposure sources (Hughes Michael 2006, Kile Molly et al. 2009 (Rausser et al. 2021)7 Lee et al. 2000b)) the dominant cellular site of oxidative phosphorylation and ROS generation(Meyer et al. 2017).mtDNA is a prime victim of oxidative stress (Yakes &Van Houten 1997).mtDNAcn in most somatic cells ranges from 100 to 10,000(Castellani et al. 2020b, Rausser et al. 2021).Mitochondria undergo fusion to complement mtDNA lesions via the transfer of nucleoids(Chen et al. 2010).Generally, the quantity of mtDNAcn within a cell is strictly governed to keep the cell in good condition(Attardi &Schatz 1988).Emerging epidemiology evidence has linked variation in mtDNAcn, either elevated or lower levels, to age-related disease(Castellani et al. 2020b, Hägg et al. 2021).Several epidemiological surveys have revealed that toxic exposures during early life were linked to changes in cord blood mtDNAcn.Increased cord blood mtDNAcn was found to be related to prenatal exposure to β-hexachlorocyclohexane(Vriens etal.2017),aluminum(Liuetal. 2019), manganese (Kupsco et al. 2019), lead, and rare earth elements (gadolinium, dysprosium, erbium, and praseodymium) (Liu et al. 2020).Other surveys have shown that declined mtDNAcn was related to maternal smoking (Janssen et al. 2017), maternal lifetime stress (Brunst et al. 2017), prenatal exposure to thallium (Wu et al. 2019), arsenic (Song et al. 2020), magnesium (Smith Anna et al. 2021), air pollutants (PM 2.5 , PM 10 , and NO 2 ) (Brunst et al. 2018, Clemente Diana et al. 2016, Hu et al. 2020, Janssen Bram et al. 2012), benzotriazoles(Chen et al. 2020).It's comprehensible that the tendency of mtDNAcn changes varies from various pollutants.Additionally, ndings for one exposure weren't consistent.Therefore, current epidemiological surveys about the relationships of prenatal exposures with alterations in child mtDNAcn couldn't draw rm agreements.This discordance in effects might be partly ascribed to the discrepancies in the characteristics of the study participants, types and levels of exposures, different tissues used to determine mtDNAcn (cord blood vs. placenta tissue), and duration of exposure.Limited research has examined the relationships of maternal arsenic metabolite concentrations with cord blood mtDNAcn.In this study, we observed positive (urinary DMA and TAs) associations with cord blood mtDNAcn.Prior surveys have shown that people in arsenic-exposed areas have higher mtDNAcn(Ameer etal.2016,Louetal.2022,Sanyaletal.2018).To our knowledge, only one study in Belgium has shown that cord blood TAs was linked to an elevated placental mtDNAcn (Vriens et al. 2017).Increased mtDNAcn manifests enhanced mitochondrial biogenesis resulting in cell aberrant proliferation (Lee et al. 2011, Malik &Czajka 2013, Wei &Lee 2002).Previous experimental surveys have displayed that low-level arsenic exposure enhanced mitochondrial biogenesis (Lee et al. 2011, Lou et al. 2022).Lee et al. found that primary keratinocytes treated with arsenic at concentrations lower than 1.0 µmol/L exhibited increased cell proliferation and mitochondrial biogenesis (Lee et al. 2011).Luo et al. found mtDNAcn in Human hepatocyte L-02 cells treated with a low dose of iAs 3+ (0.2 µmol/L) increased by more than 50% (Lou et al. 2022).Some surveys have reported that mtDNAcn increases with ROS to compensate for mtDNA lesions (Lee et al. 2000a, Liu et al. 2006,Wei et al. 2001).As a noted ROS stimulus, arsenic induces several types of ROS production, like peroxyl radicals, superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide(Banerjee et al. 2011, Lynn et al. 2000).Little research has documented arsenite's capability to promote mitochondrial ROS generation(Liu et al. 2005, Partridge et al. 2007).When ROS damaged mtDNA, reinforced mitochondrial biogenesis could increase mtDNAcn to make amends for mitochondrial malfunction(Giordano et al. 2014, Lee et al. 2000b).Nevertheless, excessive ROS might bring about declined mtDNA synthesis(Hou et al. 2010).Maintaining mtDNAcn in response to ROS induced by different pollutants remains unclear.Further surveys based on prospective cohorts and ingeniously-designed toxicological surveys are needed to elucidate the toxic mechanism for arsenic species.Our ndings also showed a possibly enhanced susceptibility to arsenic species exposure in the 3rd trimester.Probable explanations might be variations in arsenic metabolite concentrations across pregnancy or chronic arsenic toxicity in mitochondria(Partridge et al. 2007).More studies are warranted to determine possible windows of prenatal exposure to arsenic species.So far, this is the rst survey on connections between maternal arsenic species and cord blood mtDNAcn, using repeated measurements.Notwithstanding, some disadvantages couldn't be ignored.First, it wasn't possible to eliminate the residual confounding by other unmeasured factors, like the lack of cell-type data for cord blood mtDNAcn.Blood samples used to quantify mtDNAcn consist of different cell types, and mtDNAcn in diverse immune cells vary (Hurtado-Roca et al. 2016).Equally, mtDNAcn is estimated according to genomic DNA originating from the buffy coat, which is confounded by both cell type distributions and contaminating platelets(Rausser et al. 2021).The lack of data on platelet count in the cord blood samples might result in the overestimation of leukocyte mtDNAcn quanti cation in consideration of high levels of mtDNA, not nuclear DNA, in platelets (Hurtado-Roca et al. 2016, Urata et al.